Probiotics: facts and myths.

In recent years there has been a significant upsurge in research on the characterisation and verification of the potential health benefits associated with the use of probiotics. In addition, the market for probiotics continues to expand exponentially as consumers (mostly healthy individuals) rely on health claims made by manufacturers to make their choices. This review appraises the available evidence for and against the health claims associated with probiotics. The use of probiotics in promoting gastrointestinal health and immunity, and their use in the prevention of urogenital infections, allergies and cancer are reviewed. Furthermore, issues surrounding the use of probiotics in healthy individuals, the safety of probiotics and regulatory concerns are addressed. There is scientific evidence that specific strains of probiotic microorganisms confer health benefits on the host and are safe for human use. However, this evidence cannot be extrapolated to other strains, as these effects are strain-specific. Probiotics have potential health benefits for conditions such as gastrointestinal infections, genitourinary infections, allergies and certain bowel disorders, all of which afflict a considerable proportion of the global population. However, considerable work is still needed to confirm these potential health benefits.

The genotype of the hepatitis C virus in patients with HCV-related B cell non-Hodgkin's lymphoma.

Increasing evidence suggests that the hepatitis C virus (HCV) might be involved in the pathogenesis of B cell non-Hodgkin's lymphomas (NHL). Since several HCV genotypes are currently identifiable and might be involved in the pathogenesis of different diseases (with different severity and responsiveness to therapy), the aim of our study was to assess the prevalence of viral genotypes in a group of patients with HCV-related NHL. Among 470 consecutive patients, 42 HCV Ab-positive cases were identified. HCV RNA could be detected by reverse transcriptase-polymerase chain reaction and genotyping performed in 31 of these cases. As compared to our control group (211 healthy blood donors and patients with chronic liver disease), a striking high prevalence of genotype 2ac was detected among B cell NHL (48.4 vs 9.0%), with a relative risk of infection of 5.37 (P < 0.0001). No major differences were observed in the distribution of NHL histotypes and in the clinical features among patients with genotype 1b (the other most frequent genotype) or 2ac, a part from a trend towards a higher percentage of liver disease and a lower likelihood of response to interferon for patients with genotype 1b. The same high prevalence of genotype 2ac has been recently reported in patients with mixed cryoglobulinemia (MC), monoclonal gammopathies, B cell NHL complicating MC and autoimmune hepatitis. All these data taken together suggest that genotype 2ac might be involved in the pathogenesis of lymphoproliferative and autoimmune disorders.

Intracellular pH regulates the production of different oxygen metabolites in neutrophils: effects of organic acids produced by anaerobic bacteria.

The effects of short chain carboxylic acids (SCCA), namely succinic, butyric, and iso-butyric, on neutrophil metabolic activation were assessed. SCCA induced a significant decrease in O2.- recovery and chemiluminescent response in neutrophils activated with the diacylglycerol analog tetradecanoyl-phorbol-acetate (TPA). SCCA did not alter O2 consumption, H2O2 production, or the calorimetrically determined energy expenditure occurring during the metabolic burst. SCCA also induced a significant acidification of intracellular pH (pHi). These results are consistent with an increased divalent versus univalent O2 reduction performed by the NADPH oxidase at a more acidic intracellular pH.

Characterization of a putative new HPV genomic sequence from a cervical lesion using L1 consensus primers and restriction fragment length polymorphism.

Various methods have been proposed for HPV detection and typing. Prevalence and distribution among types have varied depending upon the methods used and the populations studied. We have applied the polymerase chain reaction (PCR) followed by a Restriction Fragment Length Polymorphism (RFLP) analysis using the MY09/MY11 primers for detection of HPV in cervicovaginal lavages obtained from 323 patients who were referred to our Clinical Department either for genital complaints or an abnormal PAP smear. We assessed (i) the prevalence of HPV and (ii) the reliability of RFLP-typing. For the latter, 35 PCR-HPV products were sequenced. HPV-DNA was detected in 40/197 (20.3%) patients with normal cytology 86/111 (77.5%) with LSIL and 11/15 (73.3%) with HSIL. HPV-16 was the most common type detected in normal cervical cytology samples (10/40, 25%), whereas HPV 16 and 18 were detected in 36/97 (37.1%) of the LSIL and HSIL patients, evidencing the presence of these high-risk HPV types not only in malignant conditions. Results obtained after partial nucleotide sequencing confirmed the results obtained by RFLP analysis. In this study, a putative new HPV fragment (GA6053) was identified. Its closest homology to other known HPV types is 73.8% to HPV-62, 73.0% to HPV-61 and 67.7% to HPV-18. The use of degenerate primers, in conjunction with RFLP, proved to be a reliable method for HPV detection and typing.

Impact of introducing quality control/quality assurance (QC/QA) guidelines in respiratory specimen processing.

OBJECTIVES: To assess the impact of the introduction of a new quality control/quality assurance (QA/QC) protocol on the processing and reporting of respiratory specimens. METHODS: After implementation of guidelines for processing respiratory specimens, an investigation was carried out over a six-month period on 200 specimens, 105 sputa, and 95 deep tracheal aspirates (DTAs), assessed blindly by two independent investigators. Data regarding disagreement were arranged into two subgroups. A minor disagreement was defined as a difference in the two assessments of < 10 or 10-25 for white blood cell (WBC) or squamous epithelial cell (SEC) counts. A major disagreement was defined as one assessor reporting < 10 and the other > 25 for either WBC count or SEC count, or one assessor reporting the specimen as non-assessable, or both assessors having a minor disagreement in both the WBC count and the SEC count. RESULTS: Agreement was obtained on 111 samples. For 45 specimens, a major disagreement was documented, and in 44 cases, a minor disagreement was recorded, WBC being the most common cause of divergence. Data for sputa and DTAs were examined separately: of 45 major disagreements, 64.4% were observed for DTAs, while minor disagreements were recorded mostly for sputa. The role of the settings in which samples were taken in affecting quality was studied. Among the 105 sputa, 27 were from Community Health Centers (CHCs) and 78 from hospitalized patients. Agreement between the two observers was obtained in 48.1% of CHC cases versus 60.2% of hospital samples. To investigate how many of the rejected samples presented WBC > 25 suggestive of infection, we looked at the 107 samples rejected during the six-month period, grouped according to the suspected diagnosis. The highest number of rejected samples falls in the category of unrelated (non-respiratory) diagnosis, and clinical suspicion is not helpful in Gram stain interpretation. The annual saving (not culturing, not testing, and not treating) derived from this simple QC procedure totals about 5000 Euro. CONCLUSIONS: Standardization in microscopic screening of respiratory samples is difficult to achieve. Criteria for rejection must be adapted to local conditions after discussion with clinicians to increase their compliance with the newly introduced guidelines and to avoid sending unnecessary specimens. The effects on patient management and cost control are significant.

A highly sensitive and fast non-radioactive method for the detection of polymerase chain reaction products from Salmonella serovars, such as Salmonella typhi, in blood specimens.

A polymerase chain reaction based test was developed for the detection of Salmonella spp. in blood specimens. After amplification of a 389 bp-polymerase chain reaction product from the invA gene, a microtiter plate hybridization assay was performed. The protocol described allowed the detection of six to seven copies of the Salmonella typhi genome, as determined by serial dilutions of DNA from S. typhi. Eighteen blood specimens from artificially infected rats and 22 blood specimens from patients were analyzed to validate the method. Considering that the most frequent Salmonella serovar isolated from blood in case of bacteremia is S. typhi, the polymerase chain reaction-microtiter plate hybridization technique could be used as a novel, rapid diagnostic method for typhoid fever, particularly when standard culture assays are negative.

Evaluation of a computer-assisted method of analysing SDS-PAGE protein profiles in tracing a hospital outbreak of Serratia marcescens.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of bacterial proteins have been successfully used for taxonomical purposes. More recently this technique has been applied to epidemiological investigations in respect of various micro-organisms including Neisseria meningitidis, Staphylococcus aureus and Clostridium difficile. The main limitations of the methods so far described are lack of standardisation in extraction and separation as well as in the analysis of results. Although reproducibility in the same laboratory has been shown to be satisfactory, comparison of results among laboratories is still difficult. Moreover, assessment of differences and/or similarities among chromatograms or autoradiographs showing many bands depends upon qualitative descriptions. Interpretation of densitometric scannings is laborious and time-consuming. In this paper we present our experience of a completely standardised, fully computer-controlled procedure for SDS-PAGE (AMBIS System) in analysing 35S-methionine-labelled total proteins. The methodology proved very useful in monitoring a hospital outbreak of Serratia marcescens. It allowed us to make quantitative comparison in a shorter time as well as to handle easily a great amount of data and usefully integrate it with those obtained with other systems such as serotyping. Furthermore, when the two systems are used together, more precise information can be gained. In this epidemic, serotyping indicated the presence of two groups which would have been missed by PAGE analysis alone. Electrophoretotyping, however, focused on similarities of cellular proteins among the epidemic strains. This allowed us to distinguish them from epidemiologically unrelated strains of the same serogroup.(ABSTRACT TRUNCATED AT 250 WORDS)

Short-chain fatty acids produced by anaerobic bacteria alter the physiological responses of human neutrophils to chemotactic peptide.

The effect of some short-chain fatty acids (SCFA) produced by anaerobic bacteria, namely acetic, propionic, isobutyric, butyric, isovaleric and succinic acids, on production of light and release of lysozyme by human neutrophils exposed to chemotactic peptide fMet-Leu-Phe was investigated. A short period of incubation of neutrophils with SCFA led to marked inhibition of both granulocytic chemiluminescence and degranulation (P less than 0.001). Ultrastructural studies of neutrophils, incubated with concentrations of SCFA inhibiting the chemotactic response, chemiluminescence and release of lysozyme (30 mmol/l), effected alterations in cellular morphology with formation of protrusions of varying shape. The data reported indicate that SCFA might be regarded as important pathogenicity factors. The observed effect on neutrophils could also partially explain the ability of anaerobes to inhibit their own phagocytosis and killing as well as that of the aerobic species present in mixed infections.

Configuration of the methoxyimino group and penetration ability of cefotaxime and its structural analogues.

Among the different mechanisms by which bacteria are resistant to beta-lactam antibiotics, three are of major interest: production of beta-lactamase, modifications of the target penicillin-binding proteins (PBPs) and decreased permeability. Cefotaxime (CTX) is a recently synthesized cephalosporin derivative, active against beta-lactamase producing Gram-negative bacteria and possessing in the acylamino side chain a methoxyimino group in the syn-configuration. It has been compared for affinity to PBPs and penetration ability with its isomer possessing the same group in the anti-configuration and the corresponding demethoxyimino derivative. The anti-isomer, although resistant to beta-lactamase, is devoid of antibacterial activity (MIC for Escherichia coli higher than 500 micrograms/ml). The affinities of CTX and its analogues for the PBPs of several strains of E. coli have been determined in vivo and in vitro by competition experiments using intact cells and bacterial envelopes, respectively. Only minor differences in the affinity for the target PBPs were detected in vitro. However, in vivo studies proved that the 50% saturating concentrations for the PBPs were more than 100-fold higher for the anti-isomer than for CTX. The reported results suggest that a very simple structural modification of the CTX molecule greatly decreases the penetration ability of the antibiotic through the outer cell layers, thus dramatically affecting its antibacterial properties.

Antibacterial activity of the sulbactam-ampicillin combination.

The in vitro antibacterial activity of ampicillin combined with sulbactam 2:1 was evaluated on 257 aerobic Gram-positive and Gram-negative bacteria, and on 174 anaerobic bacteria from isolated hospital strains by evaluating the minimum inhibitory concentration (MIC). The results obtained show a synergic effect which was able to significantly decrease the MIC90 of the tested beta-lactamase producing bacteria. Among the Gram-positive aerobic strains, we underline the efficacy of sulbactam/ampicillin on resistant Staphylococci, including those methicillin-resistant. The activity on the Gram-negative strains was particularly evident against enterobacteria and haemophilus. The combination of sulbactam and ampicillin provides increased activity against anaerobic strains, including Bacteroides fragilis.

Detection of HPV-DNA in semen, urine and urethral samples by dot blot and PCR.

Papillomavirus infection in women is associated with the development of carcinoma or squamous intraepithelial lesions (SIL). Limited information is available for men. Seventy asymptomatic male partners of HPV-DNA positive women were studied. Exfoliated cells collected using urethral swabs urine and semen were examined for HPV-DNA using Dot blot and PCR. On exfoliated cells collected using a urethral swab, 89% of the samples were inadequate on Dot blot, and 87% on PCR respectively. Using urine, 36% turned out to be inadequate on Dot blot, 21% on PCR. Using semen all of the 70 samples were satisfactory for both systems. Semen is thus the best material for analysis. The occurrence of HPV-DNA in urine in urine is less frequent than that in semen. Urethral swabs seem to represent the least reliable material. Carriage of human papillomaviruses is frequent in apparently healthy parternrs of HPV infected women.

Characterization of mesophilic Aeromonas from clinical specimens by computerized analysis of SDS-PAGE protein profiles and by enzymatic activity.

Mesophilic Aeromonas (Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae) have recently been considered important aetiological agents of human diseases, mainly gastrointestinal infections. Although several findings have pointed out the significance of this group of microorganisms as enteric pathogens and suggested the presence of virulence factors, epidemiological and clinical studies are limited by the difficulty of correctly identifying mesophilic Aeromonas at the species level. SDS-PAGE of radiolabelled total protein profiles and bacterial enzymatic activities were used to type 31 clinical isolates (6 A. hydrophila, 7 A. sobria and 18 A. caviae) from patients with gastroenteritis and from healthy controls. Analysis of SDS-PAGE protein patterns, reinforced by the UPGMA-grouping system (AMBIS software) provided a good characterization of A. caviae strains as a homogeneous group of microorganisms, possessing significant differences from the other two species of mesophilic Aeromonas, in good agreement with biochemical and enzymatic tests. Data obtained in analyzing A. sobria protein profiles clearly showed two groups, with a correlation coefficient (CC) = 0.70, which in our experience is a doubtful value for assigning two strains to the same species. Strains biochemically identified as A. hydrophila showed a CC = 0.64, which is equally not acceptable for species assignment. Inter-species comparison highlighted this heterogeneity, showing two mixed subgroups, both containing strains that were assigned to A. sobria and A. hydrophila species on the basis of biochemical features.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmid loss from gram-negative bacteria exposed to subinhibitory concentrations of beta-lactam drugs and azithromycin.

The stability of F'lac, pW101 and pHSG298 in Escherichia coli K12 exposed to subinhibitory concentrations of beta-lactam antibiotics, amikacin and tetracycline was studied. High molecular weight low copy plasmids (F'lac and pW101) were eliminated from bacteria treated with PBP-3 binding molecules, while a low molecular weight high copy extrachromosomal element (pHSG298) was not. None of the carbapenem antibiotics, mecillinam, amikacin or tetracycline promoted high rate plasmid loss from their hosts. Under the same conditions, plasmid-mediated ampicillin-resistance due to beta-lactamase production was also lost from F'lacTn1-carrying bacteria. In contrast, the high copy R6K plasmid was stably inherited in their hosts with the exception of those organisms treated with cefixime. When the same experiments were performed with a Klebsiella pneumoniae strain induced to form filaments by azithromycin at sub-MICs, F'lacTn1 and pW101 loss was detected, while pHSG298 was stably inherited. These results confirm previous observations that plasmid stability is correlated with cell shape and that recovery is more easily achieved when bacteria undergo an unbalanced division resulting in cell filamentation.

Analysis of amniotic fluid proteins by isoelectrofocusing (IEF).

Among the most recent methods for investigation of proteins in biological fluids SDS Polyacrylamide-gel-electrophoresis and Isoelectrofocusing (IEF) have recently been introduced into laboratory practice. The present investigation has been performed on 20 samples of amniotic fluid obtained during normal pregnancies in the first and in the third trimester. The obtained results suggest that IEF analysis seems to have a selective advantage in allowing the separation of bands which can not be easily recognized with SDS electrophoresis. These bands detected by IEF and present in amniotic fluid during late pregnancy seem to be related to some low molecular weight lipoprotein fractions and we suggest that they might be used as a possible marker for monitoring fetal lung maturation. In conclusion we think that it would be of great interest to evaluate the usefulness of IEF analysis in examining A.F. obtained during pregnancies complicated by infections.

Rapid diagnosis of Mycobacterium tuberculosis by multiplex polymerase chain reaction from clinical specimens.

An essential element in the control of tuberculosis is the rapid, sensitive and specific identification of the causative agent. Until now, screening and diagnosis are largely based on clinical signs, radiological examination, tuberculin tests, sputum examination under the microscope, or culture for mycobacteria. Tuberculin tests lack specificity and only give an indication of previous exposure to mycobacteria. Direct microscopic examination of sputum is neither specific nor sensitive enough, and mycobacterial isolation is time-consuming. As an alternative to these classical methods, new nucleic acid-based technologies show promise as a more rapid, sensitive, and specific means of identification of mycobacteria. Two commercial standardized nucleic acid-based amplification techniques have been reported to yield reliable results within 5 to 7 hrs. Roche Amplicor MTB (Roche Diagnostic System, Somerville, N.J.) and Gen-Probe AMTB (Gen-Probe Inc., San Diego, Calif.). The amplified target is part of the 16S rRNA gene which is common to all the mycobacteria. An attempt has been made to describe the use of the target DNA, SenX3-RegX3, in a multiplex PCR to detect and differentiate M. tuberculosis from other mycobacteria directly from clinical specimens.

[The therapy of odontogenic abscesses. Pharmacological experimentation with lincomycin or amoxicillin]. / Terapia degli ascessi odontogeni. Sperimentazione farmacologica con lincomicina o amoxicillina.

A clinical and microbiological study was carried out to assess the therapeutic efficacy of two different antibiotics, lincomycin and amoxicillin, in the treatment of patients suffering from odontogenic abscesses. Microbiological analyses revealed that the majority of infections were supported by mixed aerobic and anaerobic bacterial flora. The assessment of clinical parameters clearly showed that patients receiving pharmacological treatment with lincomycin achieved a more rapid and efficacious recovery from disease in comparison to patients treated with amoxicillin.